Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus.

Two RNase H (RNA-DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) activities separable by Sephadex G-100 gel filtration were identified in lysates of Moloney murine sarcoma-leukemia virus (MSV). The larger enzyme, which we have called RNase H-I, represented about 10% of the RNase H activity in the virion. RNase H-I (i) copurified with RNA-directed DNA polymerase from the virus, (ii) had a sedimentation coefficient of 4.4S (corresponds to an apparent mol wt of 70,000), (iii) required Mn-2+ (2 mM optimum) for activity with a [3-h]poly(A)-poly(dT) substrate, (iv) eluted from phosphocellulose at 0.2 M KC1, and (v) degraded [3-H]poly(A)-poly(dT) and [3-H]poly(C)-poly(dG) at approximately equal rates. The smaller enzyme, designated RNase H-II, which represented the majority of the RNase H activity in the virus preparation, was shown to be different since it (i) had no detectable, associated DNA polymerase activity, (ii) had a sedmimentation coefficient of 2.6S (corresponds to an apparent mol wt of 30,000), (iii) preferred Mg-2+ (10 to 15 mM optimum) over Mn-2+ (5 to 10 mM optimum) 2.5-fold for the degradation of [3-H]poly(A)-poly(dT), and (iv) degraded [3-H]poly(A)-poly(dT) 6 and 60 times faster than [3-H]poly(C)-poly(dG) in the presence of Mn-2+ and Mg-2+, respectively. Moloney MSV DNA polymerase (RNase H-I), purified by Sephadex G-100 gel filtration followed by phosphocellulose, poly(A)-oligo(dT)-cellulose, and DEAE-cellulose chromatography, transcribed heteropolymeric regions of avian myeloblastosis virus 70S RNA at a rate comparable to avian myeloblastosis virus DNA polymerase purified by the same procedure.     

Mechanistic Independence of Avian Myeloblastosis Virus DNA Polymerase and Ribonuclease H

Differential inhibition conditions were established for the DNA polymerase and RNase H activities of avian myeloblastosis virus (AMV) with ether-disrupted AMV and a purified enzyme preparation. The RNase H activity of ether-disrupted AMV with (rA)(n).(dT)(n) and (rA)(n).(dT)(11) as substrates was inhibited 80 to 100% by preincubation with NaF at a final reaction concentration of 27 to 30 mM. Under these conditions, the DNA polymerase activity was inhibited only 0 to 20%. Similar inhibitions were found with exogenous Rous sarcoma virus 35S and 70S RNA.DNA hybrid and phiX174 DNA.RNA hybrid as substrates. Studies were also performed with a purified enzyme preparation, in which the two activities essentially co-purified. The RNase H activity was inhibited >80% by 150 mM KCl with three different hybrid substrates, whereas the DNA polymerase activity was uninhibited. The DNA polymerase was completely inactivated by heat denaturation at 41 C or by omission of the deoxytriphosphates from the reaction mixture; the RNase H remained active. These differential inhibition conditions were used to compare the size of the DNA product synthesized with and without simultaneous RNase H action and to examine the effect of inhibition of the DNA polymerase on the size of the RNase H products. The size of the products of one activity was not affected by inhibition of the other activity. These results suggest that the AMV DNA polymerase and RNase H are not coupled mechanistically.     

Assay of hybrid ribonuclease using a membrane filter-immobilized synthetic hybrid: application to the human leukemic cell

A method for assaying hybrid ribonuclease has been devised which utilizes as substrate the synthetic hybrid [3H]polyriboadenylic acid [poly(rA)]:polydeoxythymidylic acid [poly(dT)] immobilized on the solid matrix of nitrocellulose filters. The hybridization on filter of [3H]poly(rA) to poly(dT) has been explored in terms of efficacy of the process and the response of the product to RNase H. A pulse of uv irradiation of poly(dT) while in dry state on the filter increased its firm binding to the filter in a concentration-dependent manner, resulting in a concomitant increase of the yield of hybrid formation. The filter-immobilized hybrid was 95% resistant to RNase A but sensitive to RNase H. When stored in toluene in the cold the hybrid maintained its stability for over 6 months, as judged by its resistance to RNase A. The method offers a number of advantages over assays that use solution hybrids as substrates and was readily applicable in the screening of leukemic patients, in the leukocytes of which it has demonstrated increased RNase H levels.     

Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties.

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.     

Selective inhibition of RNase H by dextran

Ordinarily, ribonuclease H hydrolyzes poly(rA) . poly(dT) and phiX174DNA-RNA at equal rates. Here we show that in the presence of dextran, the degradation of poly(rA) . poly(dT) is inhibited, while that of phi 174DNA-RNA is not. A similar inhibition by sucrose is found to be due to trace contamination of dextran in the sucrose. Ribose, deoxyribose, and a number of other saccharides fail to inhibit RNase H. In experiments where the two substrates are presented in the presence of the inhibitor, the kinetics indicates that both molecules are recognized by the enzyme, but only the phi X174DNA-RNA is degraded. That is, dextran does not interfere with the recognition site, but rather blocks hydrolysis. It is proposed that the ability of dextran to confer selectivity toward different substrates reveals a potential regulatory mechanism for RNase H activity which may represent a control step in the initiation of DNA synthesis.     

Kinetic analysis of Escherichia coli RNase H using DNA-RNA-DNA/DNA substrates.

The kinetic properties of Escherichia coli ribonuclease H (RNase H) were investigated using oligonucleotide substrates that consist of a short stretch of RNA, flanked on either side by DNA (DNA-RNA-DNA). In the presence of a complementary DNA strand, RNase H cleavage is restricted to the short ribonucleotide stretch of the DNA/RNA heteroduplex. The DNA-RNA-DNA substrate utilized for kinetic studies: (formula; see text) is cleaved at a single site (decreases) in the presence of a complementary DNA strand, to generate (dT)7-(rA)2-OH and p-(rA)2-(dT)9. Anion exchange high performance liquid chromatography was used to separate and quantitate the cleavage products. Under these conditions, RNase H-specific and nonspecific degradation products could be resolved. Kinetic parameters were measured under conditions of 100% hybrid formation (1.2-1.5 molar excess of complementary DNA, T much less than Tm). A linear double reciprocal plot was obtained, yielding a Km of 4.2 microM and a turnover number of 7.1 cleavages per s per RNase H monomer. The kinetic properties of substrate analogs containing varying lengths of RNA (n = 3-5) and 2'-O-methyl modifications were also investigated. Maximal turnover was observed with DNA-RNA-DNA substrates containing a minimum of four RNA residues. Kcat for the rA3 derivative was decreased by more than 100-fold. The Km appeared to decrease with the size of the internal RNA stretch (n = 3-5). No significant difference in turnover number of Km was observed when the flanking DNA was replaced with 2'-O-methyl RNA, suggesting that RNase H does not interact with this region of the heteroduplex.     

Inhibition of RNA-dependent DNA polymerase activity of oncornaviruses by caffeine.

Caffeine was found to inhibit RNA-dependent DNA polymerase activity of Rauscher leukemia virus when endogenous viral RNA and poly(rA)·(dT)_12–18 were used as templates. Similar results were also obtained with purified RNA-dependent DNA polymerase (deoxynucleoside triphosphate; DNA nucleotidyl transferase; EC 2.7.7.7) from avian myeloblastosis virus (AMV) utilizing 70S and 35S RNA of AMV, poly(rA)·(dT)_12–18, globin mRNA and activated calf thymus DNA as templates. The “caffeine effect” was evident only when it was present during the initiation of polymerization reaction. Increasing the template concentration in the reaction mixture partly reversed the effect of caffeine. Of the analogs of caffeine tested, only theophylline inhibited AMV DNA polymerase, whereas aminophylline showed no effect.     

Replication of poly dA and poly rA by a Drosophila DNA polymerase

The activity of a 7.3S-8.3S Drosophila DNA polymerase was characterized in detail using poly dA.p(dT)[unk] and poly rA.p(dT)[unk]. With poly dA.p(dT)[unk], Mg(2+) ion was the preferred divalent cation, and enzyme activity was inhibited by K(+) ion and by spermidine. With poly rA.p(dT)[unk], Mn(2+) ion was the preferred divalent cation and enzyme activity was stimulated by K(+) ion and by spermidine. The dependence of enzyme activity on the concentration of primer-template and on the ratio of primer to template was the same in both reactions. The two enzyme activities were identically inhibited by N-ethylmaleimide. Poly dA was replicated extensively and poly rA was replicated partially. The activation energy for poly dA replication was twice that for poly rA replication. Enzyme activity with poly dA.p(dT)[unk] was more stable to thermal inactivation than was enzyme activity with poly rA.p(dT)[unk]. These studies suggest that the same enzyme responds to both the deoxy- and the ribohomopolymer template but that the mechanisms of replication may be different.     

A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.